Exciting recent genetic studies implicate the scaffold protein SH2B1 in human disease. Human mutations in SH2B1 are associated with severe early onset obesity with disproportionately severe insulin resistance, impaired brain development, and, in some cases, short stature. This phenotype (strikingly similar to that of SH2B1-/- mice) is consistent with SH2B1 being a signaling molecule for multiple ligands, including growth hormone (GH), leptin, and insulin. SH2B1 is recruited to JAK2 and is a critical component in GH regulation of the actin cytoskeleton, a prerequisite for cellular proliferation, differentiation and migration. However, the mechanism by which SH2B1 facilitates GH-induced changes in the actin cytoskeleton remains largely unknown. The long-term goal is to understand the mechanism by which SH2B1 contributes to the function of GH and other ligands, and to gain insight into unrecognized cellular actions of GH; new functions of SH2B1; and how scaffold proteins such as SH2B1 move highly regulated and dynamic complexes from membrane-bound receptors to multiple, often remote, sites in the cell (e.g., focal adhesions, lamellipodia, cytoplasm, nucleus). The objective of the current proposal is to understand how SH2B1 regulates assembly and movement of signaling complexes and whether dysregulation of these functions contributes to defects in cellular function and ultimately human obesity, insulin-resistance, neurologic disorders, and suppressed growth. The central hypothesis is that SH2B1 facilitates the formation and/or movement of SH2B1 signaling complexes to specific locations in the cell (e.g., pm, lamellipodia, cytoplasm, nucleus) in response to GH. These functions of SH2B1 enhance GH regulation of the actin cytoskeleton, GH-induced motility, and GH responses in the nucleus. They are critical to normal human physiology, such that their impairment due to mutation contributes to the dramatic phenotype observed in human patients. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Elucidate the mechanism by which SH2B1 reversibly localizes to the plasma membrane, cytosol and nucleus. 2) Determine the role of SH2B1 in regulating GH-induced changes in the actin cytoskeleton, cell motility, and trafficking of proteins between intracellular compartments. 3) Determine the functional consequences of mutations in SH2B1 associated with human disease. Experiments will use a combination of biochemical, immunologic, cell biology, and imaging techniques standard in the lab. State-of-the-art microscopes, photoactivable probes, proteomics, cell lines and both primary macrophages and MEFs from SH2B1-/- mice, will also be used. The concept that the nuclear localization signal of SH2B1 is a dual function motif that mediates both nuclear import and plasma membrane binding that is regulated by phosphorylation of nearby serines is innovative. The proposed research is significant because it will provide insight into the role of SH2B1 in the function of GH and the numerous other ligands (e.g., leptin, insulin, nerve growth factor) that utilize SH2B1 as a signaling protein.